Efficient synthesis of phosphotyrosine building blocks using imidazolium trifluoroacetate

Article abstract of DOI:10.1016/j.tetlet.2009.09.082

We have successfully employed imidazolium trifluoroacetate as a replacement for tetrazole for efficient synthesis of phosphotyrosine. This modification is compatible with the protecting groups commonly used in solution phase and Fmoc-solid phase peptide s

Full text of DOI:10.1016/j.tetlet.2009.09.082

Tetrahedron Letters 50 (2009) 6691–6692
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient synthesis of phosphotyrosine building blocks using
imidazolium trifluoroacetate
*
Cindy Gomez, Jianyong Chen, Shaomeng Wang
Departments of Medicinal Chemistry and Internal Medicine and Comprehensive Cancer Center, University of Michigan, 1500 East Medical Center Drive,
Ann Arbor, MI 48109, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 April 2009
Revised 14 September 2009
Accepted 15 September 2009
Available online 19 September 2009
We have successfully employed imidazolium trifluoroacetate as a replacement for tetrazole for efficient
synthesis of phosphotyrosine. This modification is compatible with the protecting groups commonly used
in solution phase and Fmoc-solid phase peptide synthesis.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Phosphotyrosine
Imidazolium trifluoroacetate
The SH2 (Src Homology 2) domain is a structurally conserved
protein domain contained within the Src oncoprotein1 and in many
other intracellular signal-transducing proteins.2 A total of 120 SH2
domains have been found in 115 proteins encoded by the human
genome.3 SH2 domains typically bind a phosphorylated-tyrosine
containing short peptide motif within a target protein and this inter-
action between proteins plays a key role in mediating signal trans-
duction in cells. In the last decade, there has been a strong
research interest in the design of small-molecule inhibitors to target
the SH2 domains as chemical probes and as potential therapeutic
agents.4 The phosphotyrosine group is necessary for high-affinity
binding to the SH2 domain and is a critical component in ligands de-
signed to target the SH2 domain.4–6 Recently, we have reported the
design of compound 2 as a conformationally constrained, phospho-
tyrosine-containing peptidomimetic to target the SH2 domain in
STAT3 (Signal Transducer and Activator of Transcription 3), based
upon the pYLPQTV sequence (compound 1) from the gp130 protein.6
For the synthesis of the phosphotyrosine group, one can start
with the phosphotyrosine O-dimethyl ester, which is commercially
available. However, the conditions necessary for the removal of the
methyl ester are stringent, often resulting in low yields. Protection
of the phosphate with groups such as t-butyl or benzyl esters is
attractive because they can be removed under mild conditions
and in high yield. Since phosphotyrosine containing these types
of protecting groups is not commercially available, there is a need
for a versatile and facile synthesis of phosphotyrosine building
blocks. One of the most commonly used phosphorylation methods
N
O
NH
O
O
O
O
NH
OH
NH
HN
HN
O
O
O
NH2
NH2
OH
O
P
OH
O
1
H2N
O
O
H
N
O
O
N
HN
O
NH
O
N
O
H
OH
O
P
OH
O
2
of tyrosine uses phosphoramidite and tetrazole.7 The tetrazole
functions both as a weak acid and as a nucleophile, activating
the phosphoramidite for phosphorylation of tyrosine.8 The proce-
dure is compatible with acid-, base- and H2-labile protecting
groups. Unfortunately, tetrazole is no longer commercially avail-
able in an effective concentration. Another drawback of tetrazole
is that it is explosive when heated.
In this Letter, we wish to report an efficient, one-pot phospho-
ramidite-mediated synthesis of phosphotyrosine using imidazoli-
um trifluoroacetate9 in place of tetrazole for the synthesis of
* Corresponding author. Tel.: +1 734 615 0362; fax: +1 734 647 9647.
E-mail address: shaomeng@umich.edu (S. Wang).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.09.082

6692
C. Gomez et al. / Tetrahedron Letters 50 (2009) 6691–6692
O
R1
N
O
R1
OMe
R2
1.imidazolium trifluoroacetate in dry THF
N
OMe
R2
O
O
2.
N P
P
OH
O
O
O
3 (
R1 = H, R2 = Cbz)
4 (R1 = H, R2 = Fmoc)
7 (
R1 = H, R2 = Cbz)
5
6
(R1 = H, R2 = Boc)
(R1 = Me, R2 = Boc)
8
(R1 = H, R2 = Fmoc)
9 (R1 = H, R2 = Boc)
10
(R1 = Me, R2 = Boc)
O
O
R1
R1
R2
N
N
OMe
OH
R2
CaCl2/NaOH or
LiOH
14% Aq. tBu-OOH, overnight
OtBu
OtBu
O
O
P
O
P
Bu
Ot
OtBu
O
11 (R1 = H, R2 = Cbz, 75%)
15 (R1 = H, R2 = Cbz, 68%)
12
13
16
17
(R1 = H, R2 = Fmoc, 60%
(R1 = H, R2 = Boc, 92%)
(R1 = H, R2 = Fmoc, 92%)
(R1 = H, R2 = Boc, 97%)
14 (R1 = Me, R2 = Boc, 78%)
18 (R1 = Me, R2 = Boc, 82%)
Scheme 1. Synthesis of phosphotyrosine utilizing imidazolium.
t-butyl ester-protected phosphotyrosine. We have utilized a pro-
tection strategy which permits the synthesized phosphotyrosine
to be readily incorporated into ligands at either its C- or N-termi-
nus. Imidazolium trifluoroacetate can be readily generated from
inexpensive reagents common in most laboratories.
ture, which was then extracted with ethyl acetate, concentrated
and finally purified via column chromatography (SiO2) using
EtOAc/DCM (20/80) to elute the pure product. In larger scale
synthesis, the crude product can be cooled to 0 °C and stirred in
a solution of aqueous Na2S2O5 for 1 h, then the reaction mixture
can be extracted with ethyl acetate and purified.
In summary, we have demonstrated that imidazolium trifluoro-
acetate is a viable replacement for tetrazole in the phosphorylation
by phosphoramidite of tyrosine. This modification is compatible
with the protecting groups commonly used in solution phase and
Fmoc-solid phase peptide synthesis.
In this optimized route (Scheme 1), imidazolium trifluoracetate
was first generated in situ from equimolar amounts of imidazole
and trifluoroacetic acid in dry THF. This salt was reacted with di-
tert-butyl-N,N-di-isopropyl phosphoramidite and then with the
protected phosphotyrosine (3–6) to give the corresponding phos-
phorylated-tyrosine (7–10). This protected phosphotyrosine (11–
14) was obtained in 60–92% yield by oxidation with 14% aqueous
t-butyl peroxide. The tyrosine carboxyl group was protected as
the methyl ester rather than tert-butyldimethylsilyl ester because
the methyl ester is more stable and amenable to long term storage,
avoiding possible deprotection of the t-butyl esters on the phos-
phate by a free carboxylic acid. To obtain the free carboxylic acid,
the methyl ester was hydrolyzed by LiOH. Methyl esters containing
Fmoc groups can be hydrolyzed with CaCl2/NaOH.10,11
Acknowledgement
C.G. is grateful for the financial support from the University of
Michigan Pharmacological Sciences Training grant.
References and notes
As an example, the procedure to generate one-pot phosphoryla-
tion of protected tyrosine (12) is detailed below: Imidazole
(4.6 equiv) was dissolved in the minimum amount of dry THF
(approximately 3–5 ml) and TFA (4.6 equiv) was added gradually
to the solution under N2. The concentrated, white slurry which
was formed was stirred under N2, at room temperature for approx-
imately 10 min. Di-t-butyl di-isopropyl phosphoramidite
(1.5 equiv) was then added dropwise to the slurry, and the reaction
was stirred under N2 for approximately 10 min. The protected
tyrosine (1.0 equiv) in dry THF was then added to the reaction over
15 min and stirred at room temperature under N2 until the tyro-
sine-starting material had been consumed, as indicated by TLC.
The reaction mixture was then cooled to 0 °C and a 14% aqueous
solution of t-butyl peroxide (2.3 equiv) was added. The tempera-
ture was then allowed to rise to room temperature and the mixture
was stirred overnight. The reaction was quenched with saturated
aqueous NaHCO3 solution. Water was added into the reaction mix-
1. Sadowski, I.; Stone, J. C.; Pawson, T. Mol. Cell. Biol. 1986, 6, 4396.
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141682 A1, 2008.
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11. Selected chemical data of compounds 12 and 16: 1H NMR data for 12 (CDCl3,
300 MHz) d 1.50 (s, 18H), 3.09–3.11 (d, J = 5.6 Hz, 2H), 3.73 (s, 3H), 4.23 (t,
J = 9.0 Hz, 1H), 4.34–4.53 (m, 2H) 4.61–4.72 (m, 1H), 5.32–5.29 (d, J = 9.0 Hz,
1H), 7.03–7.06 (d, J = 9.0 Hz, 2H), 7.14–7.17 (d, J = 8.3 Hz, 2H), 7.24–7.50 (m,
4H), 7.58–7.60 (d, J = 7.3, 2H), 7.77–7.80 (d, J = 7.41 Hz, 2H). 1H NMR data for
16 (CD3OD, 300 MHz) d 1.45 (s, 18H), 2.82–3.0 (br s, 1H), 3.15–3.26 (m, 1H),
4.07–4.23 (m, 2H), 4.26–4.42 (m, 2H), 7.04–7.06 (d, J = 6.0 Hz, 2H), 7.16–7.47
(m, 6H), 7.58–7.61 (d, J = 7.8 Hz, 2H), 7.77–7.79 (d, J = 6.9 Hz, 2H).